Use of shotgun metagenomics for the identification of protozoa in the gut microbiota of healthy individuals from worldwide populations with various industrialization levels

Abstract

Protozoa have long been considered undesirable residents of the human gut, but recent findings suggest that some of them may positively affect the gut ecosystem. To better understand the role and ecological dynamics of these commensal and potentially beneficial protozoan symbionts, we need efficient methods to detect them, as well as accurate estimates of their prevalence across human populations. Metagenomics provides such an opportunity, allowing simultaneous detection of multiple symbionts in a single analytical procedure. In this study, we collected fecal samples of 68 individuals from three Cameroonian populations with different subsistence modes and compared metagenomics-based and targeted methods of detection for two common protozoan genera: Blastocystis and Entamoeba. In addition, we analyzed our data along with publicly available fecal metagenomes from various worldwide populations to explore the prevalence and association patterns of ten protozoan genera. Regarding the detection method, microscopy was much less sensitive than metagenomics for Entamoeba, whereas qPCR was at least as sensitive as metagenomics for Blastocystis sp. However, metagenomics was more likely to detect co-colonizations by multiple subtypes. Out of the ten examined genera in 127 individuals from Cameroon, Tanzania, Peru, Italy or USA, only three (Blastocystis, Entamoeba and Enteromonas) had an overall prevalence exceeding 10%. All three genera were more common in less industrialized populations and their prevalence differed between continents and subsistence modes, albeit not in a straightforward manner. The majority (72.5%) of colonized individuals carried at least two protozoan species, indicating that mixed-species colonizations are common. In addition, we detected only positive and no negative association patterns between different protozoa. Despite the pitfalls of the metagenomic approach, ranging from the availability of good-quality sequencing data to the lack of standard analytical procedures, we demonstrated its utility in simultaneous detection of multiple protozoan genera, and especially its ability to efficiently detect mixed-species colonizations. Our study corroborates and expands prevalence results previously obtained for Blastocystis sp. and provides novel data for Entamoeba spp. and several other protozoan genera. Furthermore, it indicates that multiple protozoa are common residents of the healthy human gut worldwide.

Fig 1. Concordance between detection methods for Blastocystis sp. (overall and by subtype, N = 57) and Entamoeba spp. (N = 55) in Cameroon.

Shown are the proportions of individuals positive for both methods (MG-based and targeted approach), only one of them, or none. Blastocystis sp. (B. sp.) was assessed by MG and qPCR, while Entamoeba spp. (E. sp.) was assessed by MG and microscopy (Micro).

Fig 2. Prevalence of Blastocystis sp. and its subtypes across populations according to metagenomic analysis.

Subtypes with zero prevalence (ST6, ST7 and ST9) are not shown. Population labels are created as subsistence_country, with following abbreviations: HG = hunter-gatherers, Farm = farmers, Fish = fishers, Indstr = industrialized, Cam = Cameroon, Tanz = Tanzania, It = Italy.

Fig 3. Prevalence of Entamoeba spp. and its species across populations according to metagenomic analysis.

Species with zero prevalence (E. chattoni and E. histolytica) are not shown. The population abbreviations are the same as in Fig 2.

Fig 4. Association patterns between protozoan species in non-industrialized populations.

Only species with at least one significant correlation are shown. E. = Entamoeba.